Error message: alignment amplicon sequence to reads about crispresso2 HOT 3 CLOSED

Hi,
thank you so much for the help! It works great now :)
Best!
Dimitri

from crispresso2.

The amplicon sequence is the following: GCAGGGGGCAAAGAGAGGCCGGGGAGCAAGGAGGAGGTGGATGAAGACCGCGACGTCGATGAGTCCTCCCCCCAAGACTCCCCTCCCTCCAAGGCCTCCCCAGCCC
The gRNA is the following: GGGAGGACTCATCGACGTCG

And the fastq sample example is attached.
APOE34-MAPT1-CR-clone35-C11-DB-FR-NIM_S124_L001_R1_001.fastq.gz

from crispresso2.

Hi @dbudinger

Sorry about the confusion. I took a look at the fastq file you sent. The first read does indeed contain the amplicon, but it also contains a lot of other sequence before and after the amplicon sequence:

read: GCTTCTAGGAGACCTGCACNAGGAGGGGCCGCCGCTGAAGGGGGCAGGGGGCAAAGAGAGGCCGGGGAGCAAGGAGGAGGTGGCTGAAGACCGCGACGTCGATGAGAGCACCCCCCAAAACTCCCCTCCCTCCAAGACGTCCCCAGCCCACGATGGGAGGCCTCCCCAGACAGCCGCCAGAGAATCCACCAGCATACTGTCTCTTATACACATCTCCGAGCCCACGAGACGATGCCTTACATCTCGTATGCCGTCTTCTGCTTGAAAAAAAAAAAAAAAAACACAACGGACGACCACAGAC
amplicon:                                        GCAGGGGGCAAAGAGAGGCCGGGGAGCAAGGAGGAGGTGGATGAAGACCGCGACGTCGATGAGTCCTCCCCCCAAGACTCCCCTCCCTCCAAGGCCTCCCCAGCCC

CRISPResso requires reads to align to the amplicon with a minimum 60% similarity, and because this alignment only has 106bp/301bp = 35% similarity, it is discarded.

In order to run CRISPResso, you need to trim the reads to only contain the amplicon sequence.

You can use this doing cutadapt. For example, I created a trimmed file with the following commands:

cutadapt -g GCTTCTAGGAGACCTGCACNAGGAGGGGCCGCCGCTGAAGGGG APOE34-MAPT1-CR-clone35-C11-DB-FR-NIM_S124_L001_R1_001.fastq.gz -o half.fastq.gz -e 0.2
cutadapt -a ATGGGAGGCCTCCCCAGACAGCCGCCAGAGAATCCACCAGCATACTGTCTCTTATACACATCTCCGAGCCCACGAGACGATGCCTTACATCTCGTA -e 0.2 half.fastq.gz -o trimmed.fastq.gz

trimmed.fastq.gz

This runs successfully and produces the following output.

Let me know if you'd like me to trim other files for you.

from crispresso2.